Instrument setup system for a fluorescence analyzer

Abstract

The present invention reagents and methods for setting up an instruments having a multiplicity of detector channels for analyzing a multiplicity of fluorescent dyes. The present invention is particularly applicable in the field of flow cytometry.

We claim: 
     
       1. A method for determining, in an instrument for analyzing a multiplicity of fluorescent dyes using a multiplicity of detector channels, a spillover into a secondary detection channel of a fluorescent dye that emits in a primary detection channel; wherein the method comprises:
 a) determining a calibrated spillover value by measuring, with said instrument, emissions in said primary and secondary detection channels of said fluorescent dye and of a reference material, that emits in said primary and secondary detection channels, and calculating said calibrated spillover value by:
 (i) multiplying the ratio of the emissions of the fluorescent dye by the ratio of the emissions of the reference material, each measured in the primary and secondary detection channels; or 
 (ii) multiplying the ratio of the emissions measured in the primary detection channel by the ratio of the emissions measured in the secondary detection channel; 
 
 b) measuring with said instrument, using said instrument set to adjusted photodetector gain settings, emissions of said reference material in said primary detection channel to obtain a first reference value and in said secondary detection channel to obtain a second reference value; and 
 c) determining said spillover into said secondary detection channel of said fluorescent dye by multiplying the calibrated spillover value by the ratio of the first and second reference values to thus obtain a spillover value of the fluorescent dye. 
 
     
     
       2. The method of  claim 1 , wherein said reference material comprises microparticles labeled with a plurality of fluorescent dyes. 
     
     
       3. The method of  claim 1 , wherein said instrument is a flow cytometer. 
     
     
       4. A method for determining, in an instrument for analyzing a multiplicity of fluorescent dyes using a multiplicity of detector channels having adjustable photodetector gains, a spillover, specific to a final set of photodetector gain settings, into a secondary detection channel of a fluorescent dye that emits in a primary detection channel; wherein the method comprises:
 a) determining a calibrated spillover value by measuring with said instrument, using said instrument adjusted to a first set of photodetector gain settings, emissions in said primary and secondary detection channels of said fluorescent dye and of a reference material that emits in said primary and secondary detection channels, and calculating said calibrated spillover value by:
 (i) multiplying the ratio of the emissions of the fluorescent dye by the ratio of the emissions of the reference material, each measured in the primary and secondary detection channels; or 
 (ii) multiplying the ratio of the emissions measured in the primary detection channel by the ratio of the emissions measured in the secondary detection channel; 
 
 b) measuring with said instrument, using said instrument adjusted to said final set of photodetector gain settings, emissions of said reference material in said primary detection channel to obtain a first reference value and in said secondary detection channel to obtain a second reference value; and 
 c) determining said spillover into said secondary detection channel of said fluorescent dye by multiplying the calibrated spillover value by the ratio of the first and second reference values to thus obtain a spillover value of the fluorescent dye. 
 
     
     
       5. The method of  claim 4 , wherein said first set of photodetector gain settings and said final set of photodetector gain settings are different. 
     
     
       6. The method of  claim 4 , wherein said measuring emissions of said fluorescent dye is carried out by measuring emissions of a fluorescence-matched standard in said primary and secondary detection channels. 
     
     
       7. The method of  claim 6 , wherein said fluorescence-matched standard comprises particles labeled with said fluorescent dye. 
     
     
       8. The method of  claim 7 , wherein said fluorescence-matched standard comprises hard-dyed particles labeled with said fluorescent dye. 
     
     
       9. The method of  claim 6 , wherein said fluorescence-matched standard comprises cells in a sample of blood, wherein said cells are stained with an antibody labeled with said fluorescent dye. 
     
     
       10. The method of  claim 9 , wherein said cells are CD4+ lymphocytes in a sample of fixed human blood, and said antibody binds to CD4. 
     
     
       11. The method of  claim 4 , wherein said reference material comprises microparticles labeled with a plurality of fluorescent dyes. 
     
     
       12. The method of  claim 4 , wherein said instrument is a flow cytometer. 
     
     
       13. A method for determining, in an instrument for analyzing a multiplicity of fluorescent dyes using a multiplicity of detector channels having adjustable photodetector gains, a spillover value, specific to a final set of photodetector gain settings, into a secondary detection channel of a fluorescent dye that emits in a primary detection channel,
 wherein a predetermined calibrated spillover value has been determined for said fluorescent dye by measuring with said instrument, using said instrument adjusted to a first set of photodetector gain settings, emissions in said primary and secondary detection channels of said fluorescent dye and of a reference material that emits in said primary and secondary detection channels, and calculating said predetermined calibrated spillover value by:
 (i) multiplying the ratio of the emissions of the fluorescent dye by the ratio of the emissions of the reference material, each measured in the primary and secondary detection channels; or 
 (ii) multiplying the ratio of the emissions measured in the primary detection channel by the ratio of the emissions measured in the secondary detection channel; 
 
 wherein the method comprises: 
 a) measuring with said instrument, using said instrument adjusted to said final set of photodetector gain settings, emissions of said reference material in said primary detection channel to obtain a first reference value and in said secondary detection channel to obtain a second reference value; and 
 b) determining said spillover value by multiplying the predetermined calibrated spillover value by the ratio of the first and second reference values. 
 
     
     
       14. The method of  claim 13 , wherein said first set of photodetector gain settings and said final set of photodetector gain settings are different. 
     
     
       15. The method of  claim 13 , wherein said measuring emissions of said fluorescent dye was carried out by measuring emissions of a fluorescence-matched standard in said primary and secondary detection channels. 
     
     
       16. The method of  claim 15 , wherein said fluorescence-matched standard comprises particles labeled with said fluorescent dye. 
     
     
       17. The method of  claim 16 , wherein said fluorescence-matched standard comprises hard-dyed particles labeled with said fluorescent dye. 
     
     
       18. The method of  claim 15 , wherein said fluorescence-matched standard comprises cells in a sample of blood, wherein said cells are stained with an antibody labeled with said fluorescent dye. 
     
     
       19. The method of  claim 18 , wherein said cells are CD4+ lymphocytes in a sample of fixed human blood, and said antibody binds to CD4. 
     
     
       20. The method of  claim 13 , wherein said reference material comprises microparticles labeled with a plurality of fluorescent dyes. 
     
     
       21. The method of  claim 13 , wherein said instrument is a flow cytometer. 
     
     
       22. A method for determining, in an instrument for analyzing a multiplicity of fluorescent dyes using a multiplicity of detector channels, a calibrated spillover value for determining the spillover into a secondary detection channel of a fluorescent dye that emits in a primary detection channel; wherein the method comprises:
 a) measuring with said instrument emissions in the primary and secondary detection channels of the fluorescent dye and of a reference material that emits in the primary and secondary detection channels, and 
 b) calculating the calibrated spillover value by:
 (i) multiplying the ratio of the emissions of the fluorescent dye by the ratio of the emissions of the reference material, each measured in the primary and secondary detection channels; or 
 (ii) multiplying the ratio of the emissions measured in the primary detection channel by the ratio of the emissions measured in the secondary detection channel wherein the calibrated spillover value obtained is used to determine a spillover value into the secondary channel of the fluorescent dye.